Despite 30 years of AIDS research, there is still no robust, reverse-genetic system for studying the in vivo function of human genes that influence HIV-1 replication, pathogenesis, and immunity. As the number of such genes skyrockets, including genes that determine rates of HIV-1 acquisition and disease progression among intravenous drug users, the need for such technology has never been greater. This proposal addresses technical hurdles that must be overcome before such an experimental system can be realized. RNAi transformed the functional assessment of human genes, but lack of reproducibility and inability to assess allelic variants limit utility. Mouse gene knockout technology is unsurpassed at unambiguous assignment of function to particular mammalian genes. Unfortunately, many human genes lack simple mouse orthologues, including APOBEC3G and TRIMS, two genes that restrict HIV-1 infection among intravenous drug users. Worse, HIV-1 does not replicate in mouse cells. The project proposed here will develop tools for targeted gene replacement by homologous recombination in cells of the human immune system, and for functional assessment of these modified cells within the context of an in vivo model of HIV-1 transmission, replication, immunity, and AIDS pathogenesis. Towards this end, ongoing, cutting-edge technical developments from several fields will be exploited, including human embryonic stem cells and somatic cell reprogramming, murine immune system substitution with human counterparts, lentiviral vectors that permit efficient delivery of DNA to transfection-resistant cells, and designer nucleases to stimulate homologous recombination. Development of a perpetual source of isogenic, human hematopoietic stem cells that can be genetically-modified in a controlled fashion, and used to generate an immune system within an in vivo experimental model, will permit us to draw firm conclusions concerning the function of particular human genes - or of particular alleles - in hematopoietic development, immune function, and HIV-1 replication and pathogenesis, all within a setting of substances of abuse.